A new metabolic pathway from n-dodecane to α, ω-dodecanedioic acid in a mutant of Candida tropicalis

Summary The metabolic formation of ,-dodecanedioic acid via ,-dodecanediol from n-dodecane using a mutant S76 of Candida tropicalis was studied.It was found that resting cells of S76 produce ,-dodecanediol from n-dodecane. This intermediate was identified by different analytical methods. With n-dodecanol as substrate the quantitative changes in the concentrations of ,-dodecanediol as well as other intermediates, e.g. monoacid, -hydroxy acid and ,-dioic acid produced by resting cells of S76 for different periods of time were determined. With ,-dodecanediol as the sole carbon source, quantitative changes of -hydroxy acid and ,-dioic acid produced by S76 were also recorded.The results confirm the existence of a new metabolic pathway via ,-diol in the course of ,-dioic acid formation from n-alkane in the mutant S76 of C. tropicalis.     

Formation ofα,ω-dodecanedioic acid andα,ω-tridecanedioic acid from different substrates by immobilized cells of a mutant ofCandida tropicalis

Summary The metabolic formation of either,-dodecanedioic acid or,-tridecanedioic acid from the individual n-alkane, n-alcohol, n-monoacid and,-diol with corresponding carbon chain length using K-carrageenan entrapped mutants S76 ofCandida tropicalis was studied. The immobilized cells of S76 could also directly produce-hydroxy acid and,-dioic acid from,-diol. With n-alcohol and n-monoacid as substrate, the amount of-hydroxy acid and,-dioic acid produced was also a function of the incubation time.The results demonstrated that in the immobilized cells of S76 the formation of,-dioic acid from n-alcohol can also run both via n-monoacid and via,-diol as well as in the normal cells of S76.     

Average phase difference theory and 1∶1 phase entrainment in interlimb coordination

The dynamics of coupled biological oscillators can be modeled by averaging the effects of coupling over each oscillatory cycle so that the coupling depends on the phase difference between the two oscillators and not on their specific states. Average phase difference theory claims that mode locking phenomena can be predicted by the average effects of the coupling influences. As a starting point for both empirical and theoretical investigations, Rand et al. (1988) have proposed d/dt= — K sin ), with phase-locked solutions =arcsin( /K), where is the difference between the uncoupled frequencies and K is the coupling strength. Phase-locking was evaluated in three experiments using an interlimb coordination paradigm in which a person oscillates hand-held pendulums. was controlled through length differences in the left and right pendulums. The coupled frequency _c was varied by a metronome, and scaled to the eigenfrequency _v of the coupled system K was assumed to vary inversely with _c. The results indicate that: (1) and K contribute multiplicatively to (2) =0 or = regardless of K when =0; (3) 0 or regardless of when K is large (relative to ); (4) results (1) to (3) hold identically for both in phase and antiphase coordination. The results also indicate that the relevant frequency is _c/_v rather than _c. Discussion high-lighted the significance of confirming =arcsin(/K) for more general treatments of phase-locking, such as circle map dynamics, and for the 11 phase-entrainment which characterizes biological movement systems.     

Cultivation of the agarophyte Gelidium pristoides in Algoa Bay,South Africa

Biofilms were allowed to develop on wooden slides of the River Red Gum (Eucalyptus camaldulensis Dehnh., Myrtaceae) submerged in two billabongs of south-eastern Australia. The slides were placed in the photic zone and the aphotic zone, and the biofilms sampled after eight week's growth over the summer of 1989–1990 and winter of 1990. Bacterial numbers, estimated with epifluorescence microscopy, ranged from 4–78 × 10⁶ cells cm^–2. Bacteria were more abundant in the photic zone than the aphotic zone, and more abundant in summer than winter. Fewer than 0.5% of the bacteria could be cultivated on nutrient agar plates. Concentrations of phospholipids ranged from 8–79 ng cm^–2, which corresponded to bacterial abundances of 2–17 × 10⁶ cells cm^–2. Fifty five phospholipid fatty acids (PLFA) were identified, of which 16:0 (13–29% of total PFLA) was the most common. Other abundant PFLA included 16:17c (6–28%), 18:26 (3–16%), 18:33 (4–12%), 18:19c (3–5%), 18:l7c (5–11%) and 18:0 (2–8%). Minor PLFA included 14:0, i and a 15:0, 15:0, 16:l5c, 16:113c, 18:36, 18:43, 20:46 and 20:53. The PLFA profiles of the biofilms were quite different from those of the sediments and plankton. There was a clear distinction between the PLFA profiles of summer and winter biofilms, but less evidence for unequivocal site or light-regime effects.     

Inserted sequence in the mitochondrial 23S ribosomal RNA gene of the yeast Saccharomyces cerevisiae.

Summary The sequence organization of the yeast mit-DNA region carrying the large ribosomal RNA gene and the polar locus was examined. Hybridization studies using rho^- deletion mutants and electron microscopy of the heteroduplexes formed between 23S rRNA and the appropriate restriction fragments, lead to the conclusion that the 23S rRNA¹ gene of the ⁺ strains is split by an insertion sequence of 1,000–1,100 bp. In contrast, no detactable insertion was found in the 23S rRNA gene of the ^- strains. The size and the location of the insert found in the 23S rRNA gene of the ⁺ strains appear to be identical to those of the sequence which had previously been found to characterize the difference (at the locus) between the mitDNA of the wild type strains carrying the ⁺ or ^- alleles (Jacq et al., 1977).     

Degradation pathways from n-tridecane to α, ω-tridecanedioic acid in a mutant of Candida tropicalis

Summary The metabolic formation of ,-tridecanedioic acid via n-tridecanoic acid and via ,-tridecanediol from n-tridecane in the mutant S 76 of Candida tropicalis was studied. It was found that resting cells of S 76 produced ,-tridecanediol from n-tridecane.With n-tridecanol as substrate, the ,-diol could also be detected. The mutant S 76 was able to produce ,-tridecanedioic acid using either n-tridecanol or n-tridecanoic acid as the sole carbon source. Quantitative changes in the concentration of -hydroxy tridecanoic acid and other intermediates were recorded during the formation of ,-dioic acid.The results confirm the existence of two metabolic pathways mentioned above in the course of ,-dioic acid formation from odd n-alkane in the mutant S 76 of C. tropicalis.     

Antigen selectivity characteristic of polyclonal antibodies against omega-conotoxin GVIA and N-type voltage-dependent calcium channels

The antibodies against omega-conotoxin GVIA (-CTX GVIA; N-type voltage-dependent calcium channel [VDCC] blocker) and B₁Nt (N-terminal segment [residues 1–13] of BI ₁ subunits of VDCCs) were prepared, and the selectivity for each antigen -CTX GVIA and B1Nt was investigated. For the antigen selectivity of anti–-CTX GVIA antibody against -CTX GVIA, ELISA, and immunoprecipitation were used. The reactions for ELISA and immunoprecipitation were observed except when antibody IgG purified by Protein A–Sepharose CL-4B from nonimmunized serum (purified NI-Ab) was used. The specific reactions were inhibited by 10 nM -CTX GVIA, but not by -CTX SVIB (N-type VDCC blocker), -CTX MVIIC (N- and P-type VDCC blocker), or -Aga IVA (P-type VDCC blocker). For the antigen selectivity of the anti-B1Nt antibody, analyses by ELISA, immunoprecipitation, and Western blotting were conducted. The reactions were observed except when NI-Ab was used. The ELISA and immunoprecipitation reactions were inhibited by the antigen peptide B1Nt, and the IC₅₀ values were about 1.2 × 1028 and 1.3 × 1028 M, respectively. The bands of 210 and 190 kD by Western blotting of crude membranes from chick brain were also inhibited by 1 M B1Nt. These results suggest that the antibodies prepared against -CTX GVIA and B1Nt in this work have high selectivity for their antigen. Therefore we assume that the antibodies against -CTX GVIA and B1Nt are useful tools for the analyses of the function and distribution of N-type VDCCs. The anti -CTX GVIA antibody must also be useful for the radioimmunoassay of -CTX GVIA.     

Characteristics of [125I]ω-conotoxin labeling using bifunctional cross linker DSP in crude membranes from chick brain

Characteristic of [¹²⁵I]-conotoxin (-CgTX) labeling using bifunctional cross linker (dithio bis[succinimidyl propionate]: DSP) was systematically investigated in crude membranes from chick whole brain. [¹²⁵I]-CgTX specifically labeled 216 kDa as a main and 236 kDa as a minor bands in the crude membranes under non-reduced condition, but not labeled under reduced condition. We investigated the effect of various Ca channel antagonists on [¹²⁵I]-CgTX labeling with DSP in detail, and found that there is a strong correlation between the effects of Ca channel antagonists on [¹²⁵I]-CgTX labeling of the 216 kDa band and specific [¹²⁵I]-CgTX binding. These results suggest that labeling of the 216 kDa band under non-reduced condition with [¹²⁵I]-CgTX using DSP involves the specific binding sites of [¹²⁵I]-CgTX, perhaps including one of the neuronal N-type Ca channel subunits in the crude membranes.     

Similarities of omega gliadins from<Emphasis Type="Italic"> Triticum urartu</Emphasis> to those encoded on chromosome 1A of hexaploid wheat and evidence for their post-translational processing

The -gliadins encoded on chromosome 1 of the A genome were purified from Triticum aestivum L. (2n=6x=42, AABBDD) cv. Butte86, nullisomic 1D-tetrasomic 1A of cv. Chinese Spring (CS N1DT1A), and the diploid T. urartu (2n=2x=14, AA). Reverse-phase high-performance liquid chromatography combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis of gliadin extracts from CS nullisomic-tetrasomic (NT) lines confirmed the assignment to chromosome 1A. The purified -gliadins were characterized by mass spectrometry and N-terminal sequencing. The 1A-encoded -gliadins were smaller than 1B- or 1D-encoded -gliadins. The N-terminal amino acid sequences for 1A -gliadin mature peptides were nearly identical to those for the T. urartu -gliadins and were more similar to 1D -gliadin sequences than to sequences for T. monococum -gliadins, barley C-hordeins, or rye -secalins. They diverged greatly from the N-terminal sequences for the 1B -gliadins. The data suggest that T. urartu is the A-genome donor, and that post-translational cleavage by an asparaginyl endoprotease produces those -gliadins with N-terminal sequences beginning with KEL.Communicated by J. Dvorak     

Occurrence of Curtobacterium sp. possessing ω-cyclohexyl fatty acids in soil with zinc added

A bacterial strain, Curtobacterium sp., isolated from a soil with zinc added possessed -cyclohexyl fatty acids. -Cyclohexyl undecanoic acid made up 47% of the total fatty acids; it was the most abundant fatty acid in the strain grown in tryptone medium. 12-Methyl tetradecanoic acid (23%) and 14-methyl hexadecanoic acid (22%) were also major fatty acids. The proportion of -cyclohexyl undecanoic acid increased as the pH of the medium decreased and as the culture temperature increased.The bacteria grew almost normally in zinc-enriched medium, and -cyclohexyl undecanoic acid increased with zinc concentration. Zinc added to the medium was not abundant in the cell fraction, and the ratio of increase of zinc in the cells was not so high as in the culture medium. These results suggested that -cyclohexyl fatty acids are related to the zinc tolerance of the isolated strain, and that this tolerance depends on low permeability of the membrane to zinc.     

Fatty acid content in wild type and WZSL mutant of Euglena gracilis

The effects of growth conditions on fatty acid profilewere examined in the photosynthetic wild type and inthe spontaneous non-photosynthetic WZSL mutant of theunicellular flagellate Euglena gracilis. Inthe light, the amount of polyunsaturated fatty acids(PUFAs) is higher in the wild type than in the mutant,independent of the carbon source. Among importantPUFAs, linolenic acid (18:3 3) is present inhigh amount only in wild type cells grown in the lightwith any of the tested carbon sources. The content ofother PUFAs, such as arachidonic acid (20:46), EPA (20:5 3) and DHA (22:63), is not correlated with the presence oflight or chloroplasts.The main effect of the dark in both strains is tolower the content of PUFAs and mono-unsaturated fattyacids and to increase the content of saturated fattyacids with all the carbon sources.     

Purification and characterization of hydroxycinnamoyl-coenzyme A: ω-hydroxypalmitic acid O-hydroxycinnamoyltransferase from tobacco (Nicotiana tabacum L.) cell-suspension cultures

An acyltransferase hydroxycinnamoyl-Coenzyme A: -hydroxypalmitic acid O-hydroxycinnamoyltransferase (HHT; EC 2.3.1.-), which transfers hydroxycinnamic acids from hydroxycinnamoyl-CoA thioesters to several hydroxylated fatty acid derivatives, was characterized from tobacco (Nicotiana tabacum L. cv. Xanthi nc) cell-suspension cultures. It exhibited the same properties as the enzyme previously detected in wound-healing potato tuber discs (Lotfy et al., 1994, Phytochemistry 35: 1419–1424), and especially a marked specificity for -hydroxypalmitic acid and feruloyl-CoA. It was purified 300-fold to near homogeneity from late logarithmic-phase cell suspensions. The apparent molecular mass of the native protein was 55 kDa and its isoelectric point, estimated by electrofocusing, was 4.6. The purified enzyme conjugated ferulic acid to -hydroxypalmitic acid and to 1-tetradecanol, its main lipidic substrates, suggesting that the same enzyme probably synthesizes the different esters of 1-alkanols and of -hydroxy fatty acids which are formed in vitro.Abbreviations ABA abscisic acid - IEF isoelectric focusing - HHT hydroxycinnamoyl-Coenzyme A: -hydroxypalmitic acid O-hydroxycinnamoyltransferase - pI isoelectric point     

Metabolic formation of dodecanedioic acid from n-dodecane by a mutant of Candida tropicalis

Summary These experiments studied the metabolic formation of a,-dodecanedioic acid using the mutant S 76 developed from the wild strain Candida tropicalis 1230 (capable of producing large amounts of a,-dodecanedioic acid).Our results show for the first time that 12-hydroxydodecanoic acid was excreted into the medium as a free acid.n-Dodecanol and n-dodecanoic acid were also detected in the n-dodecane medium. The mutant S 76 was able to produce a,-dodecanedioic acid using either n-dodecanol or dodecanoic acid as the sole carbon source. Quantitative cahnges in the concentrations of 12-hydroxy-dodecanoic acid and other intermediates were recorded during the formation of a,-dodecanedioic acid. S 76 was rapidly able to convert large amounts of 12-hydroxy-dodecanoic acid to a,-dodecanedioic acid.The formation of a,-dodecanedioic acid from n-dodecane via the sequence n-dodecanoln-dodecanoic acid 12-hydroxy-dodecanoic acid was confirmed.     

A neural net capable of competitive and cooperative computation

An algorithm of learning in multilayer threshold nets without feedbacks is proposed. The net is. built of threshold elements with binary inputs. During a learning process each input vector x is accompanied by a teacher's decision ({1,...,M}). The pairs (x[n], [n]) appear in successive steps independently according to some unknown stationary distribution p(x,). The problem of learning of a threshold net has been decomposed to a series of problems of learning of the threshold elements. The proposed learning algorithm of the threshold elements has a perceptron-like form. It was proven that a decision rule of the threshold net stabilizes after a finite number of steps. For definite classes {p(x, )} _* ^K of distributions p(x,), an optimal decision rule stabilizes after a finite number of steps. These classes {p(x, )} _* ^K also contain distributions describing learning processes with perturbations.     

Modulation of Na+-channels by neurotoxins produces different effects on [3H]ACh release with mobilization of distinct Ca2+-channels

1. Voltage-gated Na⁺ channels are responsible for initiation and conduction of action potentials. The arrival of an action potential at nerve terminal increases intracellular Na⁺ and Ca²⁺ concentrations. Calcium entry into neurons through voltage-dependent calcium channels is associated with a variety of intracellular processes. Scorpion neurotoxins have been used as tools to investigate mechanisms involved in neurotransmitter release. Tityustoxin (TsTX) is an -type toxin that delays Na⁺-channel inactivation. Toxin- (TiTX-) is a -type toxin that induces Na⁺-channel activation at resting potentials.2. In the present work, we describe the effects of both toxins on [³H]acetylcholine ([³H]ACh) release from rat cerebrocortical synaptosomes, in the presence or absence of the calcium channels blockers: -conotoxin-GVIA (-CgTx), 1 M; -agatoxin-IVA (-Aga), 30 nM; -conotoxin-MVIIC (-MVIIC), 1 M; or verapamil, 1M.3. TsTX evokes [³H]ACh release in a concentration-dependent manner with a gradual increase up to saturation at concentrations of 500 nM. However, release of ACh evoked by TiTX- was not linear regarding the toxin concentration. The [³H]-ACh release evoked by TsTX or TiTX- was partially inhibited by -CgTx or -Aga, and blocked with -MVIIC. Verapamil (1 M) had no effect. Tetrodotoxin blocked [³H]ACh release evoked by both toxins.4. These results show that different actions on Na⁺-channels produce different effects on [³H]ACh release with involvement of distinct presynaptic Ca²⁺-channels, which supports the idea that sodium channels may modulate neurotransmitter release.     

A novel fungal ω3-desaturase with wide substrate specificity from arachidonic acid-producing Mortierella alpina 1S-4

A filamentous fungus, Mortierella alpina 1S-4, is capable of producing not only arachidonic acid (AA; 20:4n-6) but also eicosapentaenoic acid (EPA; 20:5n-3) below a cultural temperature of 20°C. Here, we describe the isolation and characterization of a gene (maw3) that encodes a novel 3-desaturase from M. alpina 1S-4. Based on the conserved sequence information for M. alpina 1S-4 12-desaturase and Saccharomyces kluyveri 3-desaturase, the 3-desaturase gene from M. alpina 1S-4 was cloned. Homology analysis of protein databases revealed that the amino acid sequence showed 51% identity, at the highest, with M. alpina 1S-4 12-desaturase, whereas it exhibited 36% identity with Sac. kluyveri 3-desaturase. The cloned cDNA was confirmed to encode the 3-desaturase by its expression in the yeast Sac. cerevisiae. Analysis of the fatty acid composition of the yeast transformant demonstrated that 18-carbon and 20-carbon n-3 polyunsaturated fatty acids (PUFAs) were accumulated through conversion of exogenous 18-carbon and 20-carbon n-6 PUFAs. The substrate specificity of the M. alpina 1S-4 3-desaturase differs from those of the known fungal 3-desaturases from Sac. kluyveri and Saprolegnia diclina. Plant, cyanobacterial and Sac. kluyveri 3-desaturases desaturate 18-carbon n-6 PUFAs, Spr. diclina 3-desaturase desaturates 20-carbon n-6 PUFAs and Caenorhabditis elegans 3-desaturase prefers 18-carbon n-6 PUFAs as substrates rather than 20-carbon n-6 PUFAs. The substrate specificity of M. alpina 1S-4 3-desaturase is rather similar to that of C. elegans 3-desaturase, but the M. alpina 3-desaturase can more effectively convert AA into EPA when expressed in yeast. The M. alpina 1S-4 3-desaturase is the first known fungal desaturase that uses both 18-carbon and 20-carbon n-6 PUFAs as substrates.     

Characterization of<Emphasis Type="Italic"> Pseudomonas</Emphasis> sp. HK-6 cells responding to explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine)

The cellular responses of Pseudomonas sp. HK-6 to explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) have been extensively analyzed in this study. The stress shock proteins, which might contribute to enhancing the cellular resistance to the cytotoxic effect of RDX, were induced at different concentrations of RDX used as a substrate for cell culture of Pseudomonas sp. HK-6. The proteins were identified as 70-kDa DnaK and 60-kDa GroEL by SDS-PAGE and Western blot using the anti-DnaK and anti-GroEL monoclonal antibodies. The stress shock proteins induced by RDX were found to increase in proportion to the RDX concentration used for this work. Analysis of membrane fatty acids of strain HK-6 following exposure to RDX showed that the amounts of dominant lipids 16:1 7c/15:0 iso 2OH, 16:0 and 18:1 7c/9t/12t decreased substantially or were not detected in the cells exposed to RDX, while amounts of lipids 10:0 iso, 14:1 5c/5t and 16:10 methyl increased dramatically. Scanning electron microcopy analyses revealed the presence of perforations and irregular rod shapes with wrinkled surfaces for cells treated with 0.135 mM RDX for 12 h, suggesting that RDX has a substantial cytotoxic impact on cells of strain HK-6.     

Role of ω-3 fatty acid desaturases in the regulation of the level of trienoic fatty acids during leaf cell maturation

Trienoic fatty acids, namely -linolenic acid and hexadecatrienoic acid, present in leaf lipids are produced by -3 fatty acid desaturases located in the endoplasmic reticulum and plastid membranes. The changes in the level of trienoic fatty acids during leaf maturation were investigated in wild-type plants of Arabidopsis thaliana (L.) Heynh. and in the fad7 mutant deficient in the activity of a plastid -3 desaturase. The levels of trienoic fatty acids increased in 26 °C- and 15 °C-grown wild-type plants with maturation of leaves. The increase in trienoic fatty acids was mainly due to galactolipids enriched in plastid membranes. In addition, the relative levels of trienoic fatty acids in major glycerolipids, including phospholipids enriched in the endoplasmic reticulum membranes, also increased with leaf maturation. By contrast, when the fad7 mutant was grown at 26 °C, the relative levels of trienoic fatty acids in individual lipids decreased with leaf maturation. The decreases in the levels of trienoic fatty acids, however, were alleviated when the fad7 mutant was grown at 15 °C. These results suggest that the plastid -3 desaturase plays a major role in increasing the levels of trienoic fatty acids with leaf maturation.Abbreviations 163 hexadecatrienoic acid - 183 -linolenic acid - DGD digalactosyldiacylglycerol - MGD monogalactosyldiacylglycerol - PC phosphatidylcholine - PE phosphatidylethanolamine - TA trienoic fatty acid - WT wild type - -3 refers to the position of the double bond from the methyl end of a fatty acid This research was supported in part by Grants-in-Aid for Scientific research (#07251214 and #06804050 to K.I.) from the Ministry of Education, Science and Culture, Japan, and by the research grant from Shorai Foundation.     

Oscillatory reaction-diffusion equations on rings

We study the behavior of traveling waves in - systems on both homogeneous and inhomogeneous rings. The stability regions in parameter space of - waves were previously known [15, 19]; the results are extended here. We show the existence of Hopf bifurcations of traveling waves and the stability of the limit cycles born at the Hopf bifurcation for some parameter ranges. Using a Lindstedt-type perturbation scheme, we formally construct periodic solutions of the - system near a Hopf bifurcation and show that the periodic solutions superimposed on the original traveling wave have the effect of altering its overall frequency and amplitude. We also study the - system on an annulus ofvariable width, which does not possess reflection symmetry about any axis. We formally construct traveling waves on this variable-width annulus by a perturbation scheme, and find that perturbing the width of the annulus alters the amplitude and frequency of traveling waves on the domain by a small (order ²) amount. For typical parameter values, we find that the speed, frequency, and stability are unaffected by the direction of travel of the wave on the annulus, despite the rotationally asymmetric inhomogeneity. This indicates that the - system on a variable-width domain cannot account for directional preferences of traveling waves in biological systems.     

A Confirmation of 125I-ω-Conotoxin Labeled Sites in a Crude Membrane Fraction from Chick Brain as the α1 Subunit of N-Type Calcium Channels

Omega-conotoxin GVIA (-CTX), as a selective blocker for an N-type Ca²⁺ channel, has been conveniently used in many molecular biochemical and pharmacological experiments. There has been little elucidation of ¹²⁵I--CTX binding sites (mainly the 135-kDa band) in the crude membranes from chick brain, although the characteristics of specific ¹²⁵I--CTX binding and labeling sites in chick brain membranes have been investigated in our previous research. In this work, our goal is to further identify ¹²⁵I--CTX labeling sites in chick brain membranes by using anti-B1Nt antibodies (against the N-terminal segment B1Nt of N- or P-type Ca²⁺ channel ₁-subunits). The ¹²⁵I--CTX–labeled sites in chick brain membranes could be solubilized and immunoprecipitated by using an anti B1Nt antibody. The molecular weight of the immunoprecipitated protein was determined as 135 kDa, which is inconsistent with that of the specific ¹²⁵I--CTX binding protein reported previously. Moreover, the ¹²⁵I--CTX–labeled protein could be purified by the method of preparative SDS-PAGE and recognized by anti-B1Nt antibodies in Western blotting analysis. These results indicated that anti-B1Nt antibodies could truly recognize ¹²⁵I--CTX–labeled sites as the main band of 135 kDa from chick brain membranes, and the -CTX–labeled site (mainly the 135-kDa band) should be N-type Ca²⁺ channel ₁-subunits.